The standard methods for the solid phase synthesis of peptides (SPPS) on beaded resins in the normal C-to-N direction are well developed, as they must be if long peptides are to be synthesized (reviewed in M. Bodanszky et al., “Peptide Chemistry: A Practical Textbook,” Springer-Verlag, NY (2d ed., 1993) and M. Bodanszky et al., “The Practice of Peptide Synthesis,” Springer-Verlag, NY (2d ed., 1993) S. A. Kates et al., Solid-phase peptide synthesis: a practical guide. Marcel Dekker, New York (2000)). These methods are based on attaching the carboxy terminus of an amino-protected amino acid to the resin. The amino protecting group is then selectively removed. A suitably amino-protected amino acid is coupled to the resin attached nascent peptide with a suitable coupling strategy, and the amino-protecting group of the newly attached residue is removed to complete the cycle. This process is repeated until the desired peptide sequence and length is completed, and the product peptide is then cleaved from the resin, and amino acid side chain protecting groups removed using suitable methods well known in the art. Suitable amino protecting groups in common usage include Boc and Fmoc protecting groups.
Standard peptide chemistry has served as a basis for the development of combinatorial methods for the solid phase synthesis of peptide libraries of tremendous diversity (H. M. Geysen et al., Molec. Immunol., 23, 709 (1986); R. A. Houghten et al., Nature, 354, 84 (1991); K. S. Lam et al., Nature, 354, 84 (1991); reviewed in J. Ellman et al., “Combinatorial thinking in chemistry and biology” Proc. Natl. Acad. Sci. USA, 94, 2779 (1997); R. A. Houghten, “Parallel array and mixture-based synthetic combinatorial chemistry: tools for the next millennium” Annu. Rev. Pharmacol. Toxicol., 40, 273 (2000); K. S. Lam, et al., “Applications of one-bead one-compound combinatorial libraries and chemical microarrays in signal transduction research,” Acc. Chem. Res., 36, 370 (2003)). Combinatorial methods are now widely used for drug and bioactive agent discovery.
Peptide mimetics are agents closely related to peptides but with key functional group modifications tailored for specific properties and applications. Peptide mimetics are of high interest as bioactive agents and drugs, and a number of drugs and bioactive agents in current use are peptide mimetics, including ACE inhibitors (M. Harrold et al., in Foye's principles of medicinal chemistry, D. A. Williams et al., eds., Lippincott, Philadelphia (2002) at pages 533–588), HIV protease inhibitors (M. L. Sethi et al., op. cit., at 952–979), and the anti-myeloma agent Velcade (J. Adams, Drug Disc. Today, 8, 307 (2003)). Many biological processes can conceivably be targeted through suitably designed peptide mimetics, and the development of general solid-phase approaches to such agents is expected to greatly facilitate efforts to develop and refine peptide mimetics for specific applications.
Peptide mimetic combinatorial libraries, based on the normal C-to-N direction of peptide synthesis, have been described. A peptide phosphinate library has been synthesized and used to find potent and selective inhibitors of zinc metalloproteases (J. Jiracek et al., J. Biol. Chem., 270, 21701 (1995); J. Jiracek et al., J. Biol. Chem., 271, 19606 (1996); V. Dive et al., PNAS USA, 96, 4330 (1999)). A (hydroxyethyl)amine library has also been synthesized and used to find inhibitors of the prototypical aspartyl protease Cathepsin D (E. R. Kick et al., Chem. Biol., 4, 297 (1997)).
Many peptide mimetic classes of interest as drugs and bioactive agents are modified on the C-terminus, or are derived from carboxyl group reactions. Simple C-terminal peptide mimetics include peptide trifluoromethylketones (M. H. Gelb et al., Biochemistry, 12, 1813(1985); D. Rasnick, Anal. Biochem., 149, 461 (1985); B. Imperiali et al., Tetrahedron Lett., 27, 135 (1986); peptide boronic acids (D. S. Matteson et al., J. Amer. Chem. Soc., 103, 5241 (1981); C. A. Kettner et al., J. Biol. Chem., 259, 106 (1984); W. W. Bachovchin et al., Biochemistry, 27, 7689 (1988); M. P. Groziak Am. J. Ther., 8, 321 (2001); peptide hydroxamic acids (W. Zhang et al., J. Carb. Chem., 3, 151 (2001); peptide alcohols (D. S. Cafiso, Annu. Rev. Biophys. Biomol. Struct., 23, 141 (1994); J. K. Chugh et al., Biochem. Soc. Trans., 29, 565 (2001); and peptide aldehydes (H. T. Morishima, et al., J. Antibiot. (Tokyo), 23, 263 (1970); H. T. Umezawa et al., op. cit., at 259–62 (1970); R. C. Thompson, Biochemistry, 12, 47 (1973); K. L. Rock et al., Cell, 78, 761 (1994); D. Banerjee et al., Anticancer Res., 21 (6A), 3941 (2001)). Peptide mimetic classes which are accessible through carboxyl group chemistry include statine homologs (J. Marciniszyn, et al., Adv. Exp. Med. Biol., 95, 199 (1977); K. E. Rittle et al., J. Org. Chem., 47, 3016 (1982); M. H. Gelb et al., Biochemistry, 24, 8, 1813 (1985); J. A. Fehrentz et al., Biochem. Biophys. Res. Comm., 188, 873 (1992); J. A. Fehrentz op. cit., at 865; J. M. Travins et al., Org. Lett., 3, 2725 (2001); R. K. Hom et al., J. Med. Chem., 47, 158 (2004); and hydroxyethylene isosteres (G. B. Dreyer et al., Biochemistry, 31, 6646 (1992); J. J. Konvalinka et al., Eur. J. Biochem., 250, 559 (1997); M. S. Shearman et al., Biochemistry, 39, 8698 (2000); Hom et al., cited above).
Given the interest in these peptide mimetic classes, a number of approaches to C-terminally modified peptide mimetics have been described (J. Alsina et al., Biopolymers, 71, 454 (2003)). These approaches can be divided into several subcategories, including 1) attachment through the C-terminal functional group or precursor followed by standard C-to-N peptide synthesis, 2) attachment through the backbone followed by C-to-N peptide synthesis, and 3) attachment through the amino terminus followed by N-to-C (inverse) peptide synthesis (inverse solid phase peptide synthesis; ISPPS).
The first of these general approaches, based on C-terminal functional group specific attachment strategies, are limited to a specific functional group and do not allow further elaboration of the final functional group to be made on the resin, for example, to prepare additional derivatives of a solid phase attached C-terminal functional group such as an aldehyde or chloromethylketone. The second general approach does allow further reaction of the final functional group, but suffers, as does the first approach, from the limitation that the peptide chain is synthesized in the C-to-N direction, away from the C-terminal functional group. For split-pool combinatorial peptide mimetic synthesis followed by iterative deconvolution to obtain optimized agents, which is arguably one of the better approaches to combinatorial optimization, it is the last residues added to a molecule which are optimized first (D. A. Konings et al., J. Med. Chem., 40, 4386 (1997)). In both the first and second of the above cited general approaches these are the residues furthest away from the C-terminal functional group.
In contrast to the first two attachment approaches just discussed, the third approach based on ISPPS provides the C-terminus of the nascent peptide mimetic for elaboration into desired functional groups, and for further elaboration into further derivatives, and also allows the residues closest to the C-terminus to be optimized first when using split-pool/iterative deconvolution optimization strategy. There have been a number of efforts to develop effective ISPPS strategies. The first was suggested by R. L. Letsinger and M. J. Kornet, J. Amer. Chem. Soc., 85, 3045 (1963) using amino acid ethyl esters. Merrifield et al., J. Amer. Chem. Soc., 92, 1384 (1970), used protected amino acid hydrazides as building blocks for the C-terminal elongation of peptides, followed by deprotection and subsequent reaction of the hydrazide function with nitrite, allowed the next building block to be coupled by the azide method. However, the procedure is elaborate, requiring activation and coupling at low temperature with moderate yields.
Sharma et al. have described a few C-terminally modified tetrapeptide HIV-1 protease inhibitors, generated in the inverse direction. For example, see, R. P. Sharma et al., published PCT applications WO 93/05065 (18 Mar. 1993) and WO 90/05738 (31 May 1990) and Chem. Commun., 1449 (1998). Sharma's approach relies on the coupling of amino acid tri-tert-butoxysilyl (Sil) esters. More recently, A. Johannsson et al., J. Comb. Chem., 2, 496 (2000) described a modification of the method of Sharma et al. that involves the coupling of a photolabile resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. The HATU/TMP coupling method gave levels of epimerization considerably lower than those reported for other N-to-C methods, usually a ca. 5% and occasionally even below 1%. Amino acid silyl esters are however not commercially available, and are difficult to prepare, unstable to store, and unstable under peptide coupling conditions.
Alternatively, amino acid 9-fluorenylmethyl (Fm) B. Henkel et al., Liebigs Annalen-Recueil, (10), 2161 (1997)), and amino acid allyl esters (N. F. Thieriet et al., Org. Letters, 2, 1815 (2000)) have also been used for ISPPS. The Fm ester approach appears attractive considering its similarity to standard Fmoc based C-to-N SPPS, but Fm esters are not as stable as Fmoc amino acids, and Fm ester based inverse peptide synthesis apparently suffers from this limitation. The allyl ester based approach is practicable, but allyl esters are not generally available, and deprotection requires the use of 20 mol % of Pd(PPh3)4, which is a heavy metal based reagent. These strategies therefore appear less than ideal, since suitable amino acid derivatives are not generally available commercially and can be difficult to prepare, due to instability of reactants and intermediates, and to toxicity and expense of reagents.
Thus, a continuing need exists for simple and efficient methods for the inverse (N-to-C) synthesis of peptides and peptide mimetics, particularly for the synthesis of oligopeptide mimetic libraries useful for high-throughput drug screening.